Biochemistry of Infectious Prions The "protein-only" hypothesis proposes that prions are infectious proteins, which lack informational nucleic acids. Recent studies in filamentous fungi and yeast have proven this hypothesis for specific fungal proteins. Mammalian prions have also been generated in vitro. However, Koch's postulates remain unfulfilled for mammalian prions because in vitro prion propagation studies of wild type prions have used crude homogenates rather than purified proteins;and truncated, synthetic PrP molecules refolded into amyloid fibrils have thus far not caused disease when directly inoculated in non-transgenic animals. Our laboratory has recently developed an in vitro system that amplifies PrPres, the protease-resistant protein conformer associated with infectious mammalian prions, using only purified PrPC and synthetic nucleic acid molecules. Significantly, preliminary data indicate that this purified system can autocatalytically form PrPres molecules in vitro. Our specific aims will focus upon rigorously testing the "protein-only" hypothesis, and upon investigating the effects of post-translational modifications on PrPC-to-PrPres transformation in vitro. Aim 1. To generate and propagate infectious mammalian prions in a purified system in vitro. Aim 2. To determine whether strain properties of mammalian prions can be faithfully propagated by purified PrP molecules in vitro. Aim 3. To investigate the effects of PrP post-translational modifications on the efficiency and specificity of prion propagation. In summary, we propose to test rigorously the unorthodox idea that the infectious agent of diseases such as Creutzfeldt-Jakob disease (CJD) can be composed of a single protein.